Polyethylene glycol (PEG) is a versatile polymer with hydrophylic and hydrophobic properties that is widely used to conjugate with therapeutic proteins and peptides. The resultant derivatives are compounds that exhibit increased circulatory half-life, reduced immunogenicity and antigenicity, and increased resistance to proteolysis. The covalent attachment of PEG to proteins is known as PEGylation and it requires chemical modification or activation of the hydroxyl terminal group of the polymer. Several chemical groups have been used; in recent years, PEG modified with maleimide (PEG-mal) have gained popularity in the development of therapeutic proteins. PEG-mal reacts more efficiently and specifically with thiol groups in the protein or peptide of interest. Although the excess of PEG-mal is removed during purification, small amount may still be present in the final drug substance and must be considered as a product related impurity. Therefore, a method with high specificity and sensitive is desirable to evaluate not only the residual PEG-mal present in the purified PEGylated protein but also to evaluate raw materials. Since PEG-mal does not possess chemical structural features that allows its specific and sensitive detection in solution, peptide probes that contain both, a free thiol group and a fluorescent tag have been designed. This approach coupling with High Performance Liquid Chromatography (HPLC) separation significantly improve the specificity and sensitivity for free PEG-mal detection and quantitation.